A lipase is an enzyme that catalyzes the hydrolysis of triglycerides into free fatty acids and glycerol.
For utilizing lipases in an industrial scale, however, the reaction must be conducted at such a high temperature that the triglycerides to be treated are in a molten state, since most fatty oils composed of long chain saturated fatty acids are usually present as solids at ordinary temperatures. In addition, a long retention time within the enzyme reactor at a high temperature is required for carrying out a continuous enzyme reaction by employing an immobilized enzyme, so that the enzyme to be employed in such a reaction system should have a high thermostability.
As to the screening for finding thermostable lipases, it has been reported that, in particular, bacteria in the genus Pseudomonas can produce such thermostable lipases, for example, Pseudomonas mephitica var. lipolytica (Japanese Patent Publication No. 25553/1975), Pseudomonas fragi (Japanese Patent Application Kokai No. 280274/1986) and Pseudomonas fluorescens biotype I (Japanese Patent Application Kokai No. 58885/1982).
On the other hand, in recent years, genetic engineering techniques have been applied in the mass production of enzymes. Thus, a gene coding the objective enzyme is subjected to cloning and the resulting cloned DNA is ligated to yield an adequate high expression vector. The vector containing the gene of the objective enzyme is then introduced into a host cell of, such as, the bacterial strain Escherichia coli to effect a transformation. The resulting transformant is cultivated under optimum conditions for the production of the enzyme, whereby the objective enzyme can be easily produced in large amounts. Utilization of techniques of genetic engineering were attempted also in the production of lipases, wherein cloning of many lipase genes from various microorganisms had been carried out. There have been reported bacteria therefor of, for example, the genuses Staphylococcus, Pseudomonas, Bacillus and Geotrichum. Europ. Pat. Application No. 331376 A2 discloses a process for preparing a lipase by cloning the lipase gene from a bacterium of the genus Pseudomonas and utilizing this lipase gene. No mention is given, however, in this Patent Application as to a thermostable lipase.
As far as the inventors know, there is only a single report for Bacillus stearothermophilus concerning the cloning of thermostable lipase gene (Japanese Patent Application Kokai No. 228279/1987). There has not been known, in particular, any report for the cloning of a thermostable lipase gene derived from a microorganism of the genus Pseudomonas and for a process for preparing this lipase by a recombinant bacterium containing cloned thermostable lipase gene.